This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methyl transfer reactions are ubiquitous throughout nature. To date, all characterized enzymatic methyl transfer reactions can be considered nucleophilic substitution reactions: a nucleophilic methyl acceptor attacks a methyl group donor, and the methyl group is formally transferred as a carbocation. A family of radical S-adenosyl-L-methionine (SAM) methyltransferases has been identified whose members are likely to break this paradigm. These enzymes, which play roles in microbial biosynthesis, have been proposed to methylate unreactive carbon and phosphorus atoms using thevitamin B12 derivative, methylcobalamin. These are extremely difficult chemical reactions, one of which leads to the formation of the only naturally-occurring carbon-phosphorus-carbon bond. The sites of methylation are not amenable to nucleophilic activation. Therefore, the radical SAM methyltransferases apparently use an as-yet uncharacterized catalytic mechanism in which the methyl group is transferred as a radical or an anion. Through synthetic organic, biochemical, and spectroscopic approaches, the mechanisms of two radical SAM methyltransferases will be investigated. These twoenzymes will serve as model systems for the other members of the radical SAM methyltransferase family. The results of this research will ultimately be used to engineer these and related enzymes to generate novel carbon-carbon or carbon-phosphorus-carbon bonds.